Nuclear protein factor GT‐1 binds to sequence boxes II, III, II* and III* upstream of the light‐responsive pea rbcS‐3A gene. We have shown previously that box II and box III are required for expression of rbcS‐3A when redundant elements upstream of −170 (relative to the transcription start site) are removed. Here we present evidence that deletion and substitution mutations downstream of −170 which eliminate expression also decrease binding. Using a series of 2 bp substitution mutations we have defined a core of six residues (GGTTAA) within box II (GTGTGGTTAATATG) that are critical for binding. The most detrimental mutation for binding, which changes the double Gs to Cs, is sufficient to eliminate detectable expression in vivo when only 170 bp of 5′ flanking sequences are present. The simplest interpretation of these data is that GT‐1 is an activator of rbcS‐3A transcription. Footprinting experiments show that GT‐1 from both light‐grown and dark‐adapted plants binds to the same sequences in vitro. Therefore, the lack of expression of rbcS‐3A in the dark is not due to the absence of GT‐1. In our analysis of the sequence elements upstream of −170, we have mapped two additional GT‐1 sites (boxes II** and III**) between −330 and −410. The similarities and differences among the GT‐1 sites located upstream and downstream of −170 are discussed in terms of the different sequence requirements for rbcS‐3A expression during development.

Expression of the pea rbcS-3A gene, one of a family of genes encoding the small subunit of ribulosebisphosphate carboxylase [EC 4.1.1.39], is regulated by light and is restricted to chloroplast-containing cells. We analyzed the effects of light and development on rbcS-3A expression in transgenic plants. Two highly conserved sequences ("boxes" II and III) around nucleotide position -150 (relative to the transcription initiation site, + 1) are required for rbcS-3A expression. The so-dermed positive elements overlap with previously identified negative light-regulatory elements. In the case of box II, which has sequence similarity to the core enhancer motif of simian virus 40, a GG-CC transversion is sufficient to abolish expression. The effect ofmutations in boxes II and m can only be measured when sequences upstream of -170 are removed, because sequences both 5' and 3' of -170 can direct light-regulated and organ-specific expression. This implies that there is a redundancy of cis-acting elements in the 5' noncoding region of rbcS-3A. However, we show that the sequences upstream of -170 are dispensable only in the mature leaves of a green plant. In contrast, in the young, expanding leaves at the top of a green plant, as well as in seedlings, the distal elements are required for high-level expression. Therefore, redundancy is not absolute, and the requirements for rbcS-3A expression change during plant development.

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